A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies

Abstract

The neurotransmitter acetylcholine (ACh) regulates a diverse array of physiological processes throughout the body. Despite its importance, cholinergic transmission in the majority of tissues and organs remains poorly understood owing primarily to the limitations of available ACh-monitoring techniques. We developed a family of ACh sensors (GACh) based on G-protein-coupled receptors that has the sensitivity, specificity, signal-to-noise ratio, kinetics and photostability suitable for monitoring ACh signals in vitro and in vivo. GACh sensors were validated with transfection, viral and/or transgenic expression in a dozen types of neuronal and non-neuronal cells prepared from multiple animal species. In all preparations, GACh sensors selectively responded to exogenous and/or endogenous ACh with robust fluorescence signals that were captured by epifluorescence, confocal, and/or two-photon microscopy. Moreover, analysis of endogenous ACh release revealed firing-pattern-dependent release and restricted volume transmission, resolving two long-standing questions about central cholinergic transmission. Thus, GACh sensors provide a user-friendly, broadly applicable tool for monitoring cholinergic transmission underlying diverse biological processes.

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Figure 1: Development of GACh sensors.
Figure 2: Characterization of GACh sensors in cultured HEK293T cells and neurons.
Figure 3: GACh2.0 detects rapid ACh application in brain slices.
Figure 4: GACh2.0 reveals firing pattern-dependent restricted volume transmission in MEC.
Figure 5: GACh sensors reveal dynamics of endogenous ACh release in Drosophila.
Figure 6: Attention-engaging visual stimuli evoke ACh release in behaving mice.

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Acknowledgements

We thank L. Looger and colleagues for sharing their unpublished acetylcholine sensors that validated some of our results. We thank Y. Rao for generous sharing of two-photon microscopy. We are also grateful to L. Luo, S. Owen, Y. Rao, and L. Nevin for critical reading of the manuscript. We thank Z. Ye for the help in art designing. This work was supported by the National Basic Research Program of China (973 Program; grant 2015CB856402), The General Program of National Natural Science Foundation of China (project 31671118 and project 31371442), and the Junior Thousand Talents Program of China to Y.L. Additional support comes from NIH grants NS103558 (Y.L. and L.I.Z.), DC008983 (L.I.Z.), MH104227 and MH109475 (Y.Z.), MH109104 and NS022061 (L.W.R.), LH089717 (P.Q.B.), and NS053570, NS091452, NS094980, NS092548, and NS104670 (J.J.Z.). J.J.Z. is the Radboud Professor and Sir Yue-Kong Pao Chair Professor.

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Contributions

J.J.Z. and Y.L. conceived the project. M.J. did GACh screening and optimization as well as its validation in cultured neurons and IPN slices. Y.L., Y.S., Z.J.Z., and H.J. designed and performed the work on transgenic flies. M.J. and J.F. performed experiments related to calcium imaging, GPCR internalization, Tango assay, and FRET measurements. L.M. and L.Z. did in vivo imaging of GACh sensors in mouse visual cortex. M.L. supervised the imaging experiments on MHb-IPN brain slices. P.Z. and G.W. together carried out the other experiments with assistance and advice from S.W., J.C.L., N.A.G., L.W.L., J.L., Y.Z., D.A.T., L.W.R., P.Q.B., and J.J.Z. All authors contributed to data analysis. M.J., P.Z., G.W., J.J.Z., and Y.L. wrote the manuscript with input from other authors.

Corresponding authors

Correspondence to J Julius Zhu or Yulong Li.

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M.J. and Y.L. have filed patent applications whose value might be affected by this publication.

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Jing, M., Zhang, P., Wang, G. et al. A genetically encoded fluorescent acetylcholine indicator for in vitro and in vivo studies. Nat Biotechnol 36, 726–737 (2018). https://doi.org/10.1038/nbt.4184

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